Regulace exprese Ms1, sRNA z Mycobacterium smegmatis

Bacteria are exposed to various environmental conditions during their growth. They have to cope with rapid changes in temperature, lack of nutrition, etc. To survive, bacteria alter their gene expression. One type of regulation of gene expression is regulation by small RNAs (sRNAs). In bacteria, a well-studied sRNA is 6S RNA that binds to the RNA polymerase holoenzyme. However, 6S RNA has not been identified in several bacterial species. Mycobacteria are a genus that probably does not have 6S RNA. Instead, Mycobacterium smegmatis possess another sRNA - Ms1. Ms1 structurally resembles 6S RNA and indeed it was first identified as a 6S RNA structural homologue. However, Ms1 binds to RNAP devoid of any sigma factor, and, therefore, is significantly distinct from 6S RNA. This work describes regulation of expression of Ms1. DNA fragments of different length from the region upstream of the Ms1 gene were prepared. These fragments were fused to the lacZ reporter gene and their activity was tested in different growth phases and under stress. This allowed identification and characterization of the core promoter sequence and regulatory sequences that might interact with transcription factor(s). Promoter activity increased with increased length of the promoter fragment and after transition into stationary...V průběhu růstu musí bakterie čelit různým vlivům prostředí, jako jsou změna teploty, nedostatek živin a jiné. Při adaptaci na nepříznivé podmínky dochází u bakterií k přizpůsobení genové exprese. Jedním typem regulace genové exprese je regulace pomocí malých nekódujících RNA (sRNA). Dobře studovanou bakteriální sRNA je 6S RNA, která se váže na holoenzym RNA polymerázy. Avšak tato RNA byla nalezena jen u některých druhů baterií. Jedním z druhů, které pravděpodobně nemají 6S RNA jsou mykobakterie. Místo toho byla u Mycobacterium smegmatis nalezena jiná malá RNA - Ms1. Ms1 má strukturu podobnou 6S RNA a skutečně byla původně nalezena jako strukturní homolog 6S RNA. Ukázalo se však, že Ms1 se neváže na holoenzym RNA polymerázy, ale váže se na jádro RNA polymerázy. Jedná se proto o malou RNA významně odlišnou od 6S RNA. Tato práce popisuje regulaci exprese Ms1. Z regulační oblasti před genem pro Ms1 byli připraveny promotorové fragmenty různé délky. Tyto fragmenty byly vloženy před reportérový gen lacZ. Aktivita těchto promotorů byla testována v exponenciální, časné a pozdní stacionární fázi růstu a v průběhu stresu. Na základě provedených experimentů byla určena sekvence promotorového jádra a regulační oblasti, do kterých by se mohl vázat transkripční faktor. Aktivita promotoru pro Ms1 se zvýšila při...Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyPřírodovědecká fakultaFaculty of Scienc

Regulace transkripce u mykobakterií.

Bakteriálna bunka sa musí vedieť rýchlo prispôsobiť zmenám, ktorým je vystavená v prostredí. Na tieto zmeny reaguje reguláciou génovej expresie primárne na úrovni iniciácie transkripcie. Medzi formy reguláciu transkripcie môžeme zaradiť rôznu stavbu promótorových oblastí pred odlišnými skupinami génov, prítomnosť alternatívnych sigma faktorov a tiež ovplyvňovanie priebehu génovej expresie pôsobením transkripčných faktorov buď ako aktivátory, alebo ako inhibítory. V tejto práci sú popísané spôsoby regulácie transkripcie u baktérií rodu Mycobacterium, ku ktorým zahrňujeme aj patogénne baktérie M. tuberculosis a M. leprae. Baktérie tohto rodu vykazujú malú konzervovanosť promótorových oblastí, veľké množstvo sigma faktorov, veľký počet transkripčných faktorov, prítomnosť dvojkomponentových systémov a mnoho, zatiaľ málo preskúmaných, malých regulačných RNA.The bacterial cell has to be able to cope with environmental changes. Adaptation to these changes is achieved by changes in gene expression. Gene expression is regulated mostly at the level or transcription initiation. Transcription initiation depends on the sequence of promoters and is regulated by alternative sigma factors and many transcription factors acting either as activators or repressors. This work describes various ways of transcription regulation in the bacterial genus Mycobacterium that includes deathly pathogens such as M. tuberculosis and M. leprae. The typical characteristics of this genus are poorly conserved promoters, a high number of sigma and transcription factors, the presence of two-component systems and a lot of small RNAs that have not been characterized in detail so far.Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologiePřírodovědecká fakultaFaculty of Scienc

YbeY is required for ribosome small subunit assembly and tRNA processing in human mitochondria.

Mitochondria contain their own translation apparatus which enables them to produce the polypeptides encoded in their genome. The mitochondrially-encoded RNA components of the mitochondrial ribosome require various post-transcriptional processing steps. Additional protein factors are required to facilitate the biogenesis of the functional mitoribosome. We have characterized a mitochondrially-localized protein, YbeY, which interacts with the assembling mitoribosome through the small subunit. Loss of YbeY leads to a severe reduction in mitochondrial translation and a loss of cell viability, associated with less accurate mitochondrial tRNASer(AGY) processing from the primary transcript and a defect in the maturation of the mitoribosomal small subunit. Our results suggest that YbeY performs a dual, likely independent, function in mitochondria being involved in precursor RNA processing and mitoribosome biogenesis. Issue Section: Nucleic Acid Enzymes

Predicting the mutations generated by repair of Cas9-induced double-strand breaks.

The DNA mutation produced by cellular repair of a CRISPR-Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs. We performed the experiments in a range of genetic backgrounds and using alternative CRISPR-Cas9 reagents. In total, we gathered data for >109 mutational outcomes. The majority of reproducible mutations are insertions of a single base, short deletions or longer microhomology-mediated deletions. Each gRNA has an individual cell-line-dependent bias toward particular outcomes. We uncover sequence determinants of the mutations produced and use these to derive a predictor of Cas9 editing outcomes. Improved understanding of sequence repair will allow better design of gene editing experiments

CEDAR, an online resource for the reporting and exploration of complexome profiling data

Complexome profiling is an emerging ‘omics’ approach that systematically interrogates the composition of protein complexes (the complexome) of a sample, by combining biochemical separation of native protein complexes with mass-spectrometry based quantitation proteomics. The resulting fractionation profiles hold comprehensive information on the abundance and composition of the complexome, and have a high potential for reuse by experimental and computational researchers. However, the lack of a central resource that provides access to these data, reported with adequate descriptions and an analysis tool, has limited their reuse. Therefore, we established the ComplexomE profiling DAta Resource (CEDAR, www3.cmbi.umcn.nl/cedar/), an openly accessible database for depositing and exploring mass spectrometry data from complexome profiling studies. Compatibility and reusability of the data is ensured by a standardized data and reporting format containing the “minimum information required for a complexome profiling experiment” (MIACE). The data can be accessed through a user-friendly web interface, as well as programmatically using the REST API portal. Additionally, all complexome profiles available on CEDAR can be inspected directly on the website with the profile viewer tool that allows the detection of correlated profiles and inference of potential complexes. In conclusion, CEDAR is a unique, growing and invaluable resource for the study of protein complex composition and dynamics across biological systems

Regulation of expression of Ms1, a sRNA from Mycobacterium smegmatis

Bacteria are exposed to various environmental conditions during their growth. They have to cope with rapid changes in temperature, lack of nutrition, etc. To survive, bacteria alter their gene expression. One type of regulation of gene expression is regulation by small RNAs (sRNAs). In bacteria, a well-studied sRNA is 6S RNA that binds to the RNA polymerase holoenzyme. However, 6S RNA has not been identified in several bacterial species. Mycobacteria are a genus that probably does not have 6S RNA. Instead, Mycobacterium smegmatis possess another sRNA - Ms1. Ms1 structurally resembles 6S RNA and indeed it was first identified as a 6S RNA structural homologue. However, Ms1 binds to RNAP devoid of any sigma factor, and, therefore, is significantly distinct from 6S RNA. This work describes regulation of expression of Ms1. DNA fragments of different length from the region upstream of the Ms1 gene were prepared. These fragments were fused to the lacZ reporter gene and their activity was tested in different growth phases and under stress. This allowed identification and characterization of the core promoter sequence and regulatory sequences that might interact with transcription factor(s). Promoter activity increased with increased length of the promoter fragment and after transition into stationary..

Regulation of transcription in mycobacteria.

The bacterial cell has to be able to cope with environmental changes. Adaptation to these changes is achieved by changes in gene expression. Gene expression is regulated mostly at the level or transcription initiation. Transcription initiation depends on the sequence of promoters and is regulated by alternative sigma factors and many transcription factors acting either as activators or repressors. This work describes various ways of transcription regulation in the bacterial genus Mycobacterium that includes deathly pathogens such as M. tuberculosis and M. leprae. The typical characteristics of this genus are poorly conserved promoters, a high number of sigma and transcription factors, the presence of two-component systems and a lot of small RNAs that have not been characterized in detail so far

Regulation of transcription in mycobacteria.

The bacterial cell has to be able to cope with environmental changes. Adaptation to these changes is achieved by changes in gene expression. Gene expression is regulated mostly at the level or transcription initiation. Transcription initiation depends on the sequence of promoters and is regulated by alternative sigma factors and many transcription factors acting either as activators or repressors. This work describes various ways of transcription regulation in the bacterial genus Mycobacterium that includes deathly pathogens such as M. tuberculosis and M. leprae. The typical characteristics of this genus are poorly conserved promoters, a high number of sigma and transcription factors, the presence of two-component systems and a lot of small RNAs that have not been characterized in detail so far

Analysis of current views of mutism in specialized Czech and foreign literature

This diploma thesis deals with mutism from the perspective of specialized czech and foreign literature. The character of the thesis is theoretical and the thesis is divided into three parts. In the first chapter, there is mutism described based on the study of czech specialized literature as well as etiology, symptoms, diagnostics and treatment. The second chapter describes the same categories but based on the study of foreign literature. The last part is dedicated to the analysis and comparison of acquired knowledge in particular areas. The most signifiant differences can be found in the area of diagnostics and therapy of mutisms, which are more elaborated in foreign literature than in Czech literature. Some differences appear in all other areas of mutism. The learning theory is the characteristic feature of foreign literature in the field of etiology of mutism, also the social enxiety is the characteric feature in the area of symptoms and treatment; overall we can see a dominant influence of the Diagnostic and Statistical Manual of Mental Disorders. The Czech literature provides a number of different views not only on the definition of mutism, but also on the etiology and symptomatology. The diagnostics and the therapy of mutism is not further elaborated in the Czech literature

Duplexing complexome profiling with SILAC to study human respiratory chain assembly defects

Complexome Profiling (CP) combines size separation, by electrophoresis or other means, of native multimeric complexes with protein identification by mass spectrometry (MS). Peptide MS analysis of the multiple fractions in which the sample is separated, results in the creation of protein abundance profiles in function of molecular size, providing a visual output of the assembly status of a group of proteins of interest. Stable isotope labeling by amino acids in cell culture (SILAC) is an established quantitative proteomics technique that allows duplexing in the MS analysis as well as the comparison of relative protein abundances between the samples, which are processed and analyzed together. Combining SILAC and CP permitted the direct comparison of migration and abundance of the proteins present in the mitochondrial respiratory chain complexes in two different samples. This analysis, however, introduced a level of complexity in data processing for which bioinformatic tools had to be developed in order to generate the normalized protein abundance profiles. The advantages and challenges of using of this type of analysis for the characterization of two cell lines carrying pathological variants in MT-CO3 and MT-CYB is reviewed. An additional unpublished example of SILAC-CP of a cell line with an in-frame 18-bp deletion in MT-CYB is presented. In these cells, in contrast to other MT-CYB deficient models, a small proportion of complex III2 is formed and it is found associated with fully assembled complex I. This analysis also revealed a profuse accumulation of assembly intermediates containing complex III subunits UQCR10 and CYC1, as well as a profound early-stage complex IV assembly defect